1. Field of the Invention
This invention relates to selection and use of lactic acid bacteria for improved breast milk for feeding to babies.
2. Description of the Related Art
The hygiene hypothesis of allergic disease suggests that environmental changes in the industrialized world have led to reduced microbial contact at an early age and thus resulted in the growing epidemic of allergic disease such as atopic eczema, allergic rhinoconjunctivitis, and asthma. One such environmental change that has been discussed is our changed ingestion of natural microbes of various kinds due to improved hygiene, standard of living, eating habits etc. This has led to shifts in the composition of our natural gut flora (Journal of Allergy and Clinical Immunology, 2001; 108: 516-520) and also changes in the composition of the breast milk of a lactating mother. Such changes have been connected to the prevalence of allergies of various kinds. It has also been reported that infants that are the later additions to larger families have a reduced risk of allergy than their siblings that were born early (the first of the brood). This can imply that the breast milk of a mother “improves” with increasing number of pregnancies.
Human milk contains a variety of components important in the immune system, such as macrophages, immunoglobulins, or antimicrobial proteins, which are thought to protect against infection and inflammation in the gastrointestinal and respiratory tract of the milk-fed offspring. In addition, the presence of other potentially immunomodulating factors (e.g., complex oligosaccharides, growth factors, enzymes, hormones, or cytokines) has been discussed. These beneficial properties together with the high availability of nutrients and the low antigen content of human milk are the physiologic basis of the current recommendation by pediatric experts that breast milk is the best food for infants, especially those with a family history of allergies.
Thus, it is known that breast milk contains a series of cytokines and chemokines that potentially could affect the development of allergy in the infant. It has previously been reported that components that modulate allergic reactions, such as cytokines, chemokines, and adhesion molecules, are secreted in milk at various stages of lactation (S Rudloff et al, Allergy 1999, 54, 206-211). Cytokines or chemokines could be either beneficial or disadvantageous to the breast-fed infant.
It is also known that cytokines delivered by the breast milk of animals have the ability to survive passage through the GI tract of the offspring. For example, homozygous TGF-beta-1-knock out mice die of widespread inflammatory disease after weaning, presumably being saved until weaning by the transfer of maternal milk delivered TGF-beta (Kulkami A B et al., Am J Pathology 1993; 143, 3-9). The TGF-beta survives passage all the way to the colon. IL-10 probably also survives in this way and thus delivery from the breast milk allows the IL-10 to reach the GI tract and potentially induce beneficial anti-inflammatory effects.
Further, Hawkes J S et. al. (Lipids 2001 October 36:1179-81) reported that long-chain polyunsaturated fatty acids have been associated with aspects of immune regulation including cytokine production. The purpose of this study was to investigate the effect of maternal dietary supplementation with tuna oil, rich in docosahexaenoic acid (DHA), on the concentration of transforming growth factor beta 1 (TGF-beta-1) and TGF-beta-2 in breast milk. In this randomized, dietary intervention trial, mothers of term infants consumed a daily supplement of 2000 mg oil containing either placebo (n=40), 300 mg DHA (n=40), or 600 mg DHA (n=40). The DHA increase in milk and plasma was proportional to dietary DHA. There was no relationship between milk DHA status and TGF-beta-1 and TGF-beta-2 levels.
IL-10 is a well-documented and accepted anti-inflammatory cytokine. The implication is that it will have anti-inflammatory effects in the GI tract of the infant. This is good for the infant—breast milk is generally considered to be anti-inflammatory for the infant in the sense that the infant should not overreact to pathogens/infections that appear in the gut early in life. Further, animal data points to the possible benefits for the offspring of IL-10 in the milk. An Australian group looked at an animal model where the allergy to ticks was investigated. Animals which were negative to a skin prick test (SPT) for ticks had IL-10 and IL-4 production, with the IL-10 dampening the IgE (allergic response) and the animals showing no allergy. In the allergic SPT +ve (histamine) animals, only IL-4 was produced and there was no IL-10 production. Thus IgE was not lowered and the allergy prevailed. Thus IL-10 dampens the allergy-inducing effects of IgE and may prevent allergy development.
TGF-beta-2 (Transforming Growth Factor) is also a well documented growth factor/cytokine. Its sources include for example platelets that yield milligram amounts of TGF-beta/kilogram. The factor and its isoforms (see below) can also be isolated from other tissues (microgram TGF/kg) and is found predominantly in spleen and bone tissues. Human milk also contains this factor and it is synthesized also for example by macrophages (TGF-beta-1), lymphocytes (TGF-beta-1), endothelial cells (TGF-beta-1), keratinocytes (TGF-beta-2), granulosa cells (TGF-beta-2), chondrocytes (TGF-beta-1), glioblastoma cells (TGF-beta-2), leukemia cells (TGF-beta-1).
Depending upon the cell type and conditions, the secretion of TGF-beta can be induced by a number of different stimuli including steroids, retinoids, EGF (Epidermal Growth Factor), NGF, activators of lymphocytes, vitamin D3, and IL1. The synthesis of TGF-beta can be inhibited by EGF, FGF (Fibroblast Growth Factor), dexamethasone, calcium, retinoids and follicle stimulating hormone. TGF-beta also influences the expression of its own gene and this may be important in wound healing. TGF-beta exists in at least five isoforms, known as TGF-beta-1, TGF-beta-2, TGF-beta-3, TGF-beta-4, TGF-beta-5, that are not related to TGF-alpha. The amino acid sequences of these isoforms display homologies on the order of 70-80 percent. TGF-beta-1 is the prevalent form and is found almost ubiquitously while the other isoforms are expressed in a more limited spectrum of cells and tissues. Isoforms isolated from different species are evolutionarily closely conserved and have sequence identities on the order of 98 percent. Mature human, porcine, simian and bovine TGF-beta-1 are identical and differ from murine TGF-beta-1 in a single amino acid position. Human and chicken TGF-beta-1 are also identical.
It has further been reported that members of the transforming growth factor beta (TGF-beta) family are pleiotropic cytokines with key roles in tissue morphogenesis and growth (Ingman W V, Bioessays 2002 October 24:904-14). TGF-beta-1, TGF-beta-2 and TGF-beta-3 are abundant in mammalian reproductive tissues, where development and cyclic remodeling continue in post-natal and adult life. Potential roles for TGF-beta have been identified in gonad and secondary sex organ development, spermatogenesis and ovarian function, immunoregulation of pregnancy, embryo implantation and placental development.
Rautava et. al. in Journal of Pediatric Gastroenterology and Nutrition 38:378-388, April 2004, states that TGF-beta-2 and IL-10 appear to function in a synergistic fashion with TGF-beta-2 favoring the production of IL10.
Mastitis is an inflammation of the breast that is often characterized by tenderness and erythema and sometimes fever, and is related to TGF-beta-2. During mastitis, the tight junctions of mammary alveolar cells open up, and this process is accompanied by an increase in sodium, inflammatory cells, and inflammatory and immunological mediators in breast milk. Mastitis is usually unilateral, and the highest incidence is in the first several weeks of breastfeeding. In industrialized countries, mastitis has generally been considered a problem of low morbidity, as affected women are often treated by midwives and nurse practitioners. Mastitis appears to be more common than previously believed, as large, longitudinal studies that have followed lactating women in the USA, Finland, and Australia (Semba R., Annals of the New York Academy of Sciences. November 2000; 918:156-62) suggest that 20-33% of women may develop clinically apparent mastitis. The number of lactating women with sub-clinical mastitis is logically therefore even greater.
Also mastitis has recently been linked with higher human immunodeficiency virus (HIV) load in breast milk and higher risk of mother-to-child transmission of HIV. (Semba, R. D., N. Kumwenda, T. E. Taha, et al. 1999. Mastitis and immunological factors in breast milk of human immunodeficiency virus-infected women. J. Hum. Lact. 15(4): 301-306).
TGF-beta-2 in breast milk is mostly from epithelial origin even if it is synthesized by many other cells, including B- and T-cells. Therefore increased level of TGF-beta-2 could be a mediator of or an effect of a sub-clinical breast inflammation. The ratio of sodium and potassium in breast milk (Na/K ratio) is said to be a well known predictor of infection and sub-clinical breast inflammation.
Also, Kalliomaki et. al. in J Allergy Clin Immunol. 1999 December; 104(6):1251-7, has suggested that TGF-beta in colostrum may prevent the development of allergic disease during exclusive breast-feeding and promote specific IgA production in human subjects.
Further, various locations of the body of humans and other mammals are inhabited by many different species of bacteria, including a number of different species of Lactobacillus. Such bacteria many times coexist with their host giving synergistic beneficial effects of various kinds, nowadays also known to be diverse and dependent upon the actual strain of bacteria. Different lactic acid bacteria strains, for example, L. reuteri SD2112, have specific antigens either on their surface or released by the bacterium in the gastro-intestinal tract of the mother. The data in Valeur et al, AEM, 70, 1176-1181 (2004) shows that ingested L. reuteri SD2112 can affect the levels of CD4+T-helper cells in the ileum of a healthy human as one example. Such observations have also been made in other mammalian species and avians indicating this may be a fundamental signaling system between gut flora and host. Via the so-called entero-mammary link, antigens from the active strains are actively transported to the lymph regions, i.e. the Peyer's patches, beneath the epithelium of the GI tract. Antigen-specific B-cells are then activated after which they migrate from the GI tract epithelium via the circulation to other mucosal membranes in the body including the salivary and mammary glands. The expression of specific molecules on these cells is thought to direct their adhesion to these tissues. Once in the mammary gland, these immune cells then direct other processes to determine the levels of cytokines produced locally. This type of signaling via the entero-mammary link has been demonstrated in the generation of secretory IgA in breast milk and is highly likely to apply to cytokine production also.
It has earlier been suggested (Laiho et al, Pediatric Research 53:642-647, 2003) that the observed associations between nutritional and inflammatory factors in breast milk shows that it may be possible to influence the immunologic properties of breast milk by dietary intervention of the mother. The same group noted that mothers with allergic disease had a lower concentration of TGF-beta-2 in breast milk compared with those without. In their hands IL-10 was detected, only at low levels and frequency naturally in breast milk, with no difference between mothers with allergic disease or not. It was suggested that protection from allergic disease was, mainly via induction of oral tolerance for TGF-beta-2 and IL-10, and that particularly breast milk TGF-beta-2 may play a key role with respect to the prevention of allergic disease. However, Weiner H. reported in Microbes and Infection, Volume 3, Issue 11, September 2001, pages 947-954 that because regulatory T cells generated by oral antigen are triggered in an antigen-specific fashion but suppress in an antigen-nonspecific fashion, they mediate bystander suppression when they encounter the fed auto-antigen at the target organ. Thus, mucosal tolerance can be used to treat inflammatory processes that are not autoimmune in nature.
Different Lactobacillus species, including Lactobacillus reuteri, have been used in so called probiotic formulations, meaning supplying an animal, including humans, with live and beneficial microorganisms. Lactobacillus reuteri is one of the naturally occurring inhabitants of the gastrointestinal tract of animals, and is routinely found in the intestines, and occasionally in the birth channel, breast milk and mouth of healthy animals, including humans. It is known to have antibacterial activity. See for example, U.S. Pat. Nos. 5,439,678, 5,458,875, 5,534,253, 5,837,238, and 5,849,289. When L. reuteri cells are grown under anaerobic conditions in the presence of glycerol, they produce the antimicrobial substance known as reuterin (β-hydroxy-propionaldehyde).
Lactic acid bacteria have earlier also been reported to be used to prevent and treat allergies of various sort for example can the following patent/patent applications be mentioned EP 1239032 by Stadler et al regarding new recombinant strains, WO 01/37865 by Clancy et al regarding lowering the amount of IgE by lactobacilli.
It is therefore an object of the invention to provide selected lactic acid bacteria and components thereof for improved breast milk for feeding to babies, and a method of such selection. More precisely, it is an object of the invention to increase the levels of the anti-inflammatory cytokine IL-10 in the milk for reducing the risk that the feeding baby develops allergy at the same time as reducing the cause and thereby the amount of TGF-beta-2 in the milk and the risk for the lactating mother to develop mastitis.
It is therefore one object of the invention to compensate for negative changes in microbial flora by giving specifically selected strains of lactic acid bacteria to mothers, before and during breast-feeding.
It is another object of the invention to select, using the herein described method or similar distinguishing specific cytokine influence of the test strains on relevant cell types, and use such certain strains of lactic acid bacteria as dietary components for mothers that stimulates increased production of IL 10 in the breast milk at the same time as reducing the TGF-beta-2 level, indicating lower level of sub-clinical inflammation in the breast milk glands and other tissue and therefore reduced risk of mastitis. Mastitis and sub-clinical mastitis can be considered to interfere with breast feeding and thus preventing benefit to the infant that breast milk provides—the selected lactobacilli by the method of the present invention then may improve mothers health and allow them to give milk for a longer time.
It is a further object of the invention to provide products containing said strains, mutants, metabolites or components thereof, including agents for administration to animals, including humans.
Other objects and advantages will be more fully apparent from the following disclosure and appended claims.